1.0 Field of the Invention
The invention relates to compositions and methods for the cryopreservation of cellular vaccines. More specifically, the invention relates to the use of particular compositions comprising cryoprotective media that includes hydroxyethyl starch (HES) and glycerol or DMSO together with recombinant live cancer cells. The compositions can be frozen and thawed, when desired, for use as a cellular vaccine for the treatment of cancer.
2.0 Background of the Invention
At certain periods of time in a tumor's cell growth, the patient's immune system has the ability to recognize the growth as abnormal (or non-self). As a result, various methods have been developed which take advantage of the patient's own immune system to fight cancer. Exemplary methods include the use of polyclonal and monoclonal antibodies, non-specific immune system stimulants, such as cytokines, protein or peptide subunit vaccines (e.g., using antigens that are often associated with cancer cells, such as tumor-specific antigens and tumor-associated antigens), adoptive immunotherapy or cellular therapy, gene therapy, cellular vaccines, etc. See, e.g., WO 00/33871.
Cellular vaccines wherein cells (or derivatives thereof) are the therapeutic agent are currently in clinical testing for treatment of cancer. Such cellular vaccines provide advantages over isolated protein vaccines in that whole cells are the vehicle for a broad range of antigens, e.g., on the cell surface. See, for example, Dranoff et al. WO 93/06867, Gansbacher et al., WO 94/18995, Jaffee et al. WO 97/24132, Mitchell et al. WO 90/03183 and Morton et al. WO 91/06866, each of which is expressly incorporated by reference herein.
Cells for use in such cellular vaccines can be modified, e.g., to express a protein which modulates the immune response to the cell. For example, a gene encoding a cytokine or costimulatory molecule may be introduced into cells derived from, e.g., a primary tumor taken from a patient, to create recombinant cancer cells. When the cytokine or costimulatory molecule is expressed, it is capable of modulating the immune response to the cell. The recombinant cancer cells may be expanded in vitro, treated to prevent further growth and returned to the patient. Appropriate timing of administration(s) and good cell viability are required for effective treatment with a cellular vaccine. Thus, it is important to be able to store recombinant cancer cells for use at selected time points, appropriate to particular treatments. Storage and maintenance of viability are important in order to allow for transportation, to decrease the amount of cell divisions the cells undergo before use in treatment and to ensure that an adequate and reproducible dose is delivered to the patient.
Freezing of cell compositions with maintenance of viability has been the subject of considerable research. Maintenance of cell viability following freezing and thawing continues to be a challenge. In the freezing process as the liquid component of a cell is changed to a solid, ice crystals are formed and damage can occur to the cells. At least two types of damage to cells is possible when ice crystals are formed. Rapid growth of ice crystals may physically disrupt membranes and subcellular organelles and may even lyse cells. Slow growth of ice crystals may result in cellular dehydration (because of the exclusion of electrolytes from the ice crystals) and extra-cellular ice formation. See, e.g., Gorlin, (1996) Journal of Infusional Chemotherapy, 6(1):23–27.
In an attempt to minimize the effects of ice crystal formation, cells are typically frozen in medium with cryoprotectants. Cryoprotectants protect the cells during freezing in a variety of ways. Collagiative cryoprotectants penetrate the cell and decrease the osmotic gradient across membranes. Vitrifying cryoprotectants increase the glass formation of the solution thereby creating a glass wall around the cell, which prevents dehydration. Cryoprotectants can also work by inhibiting ice crystal formation. See, e.g., Gorlin, (1996) Journal of Infusional Chemotherapy, 6(1):23–27.
Different cell types vary in their permeability to water and in their sensitivity to solute concentration. Leibo et al., (1970) Cryobiology, 6(4): 315–332. As a result, different types and combinations of cryoprotectants have been found to be effective to preserve specific types of cells. For example, human bone marrow committed stem cells have been shown to be preserve by a cryoprotectant combination of dextran, glycerol, and dimethyl sulfoxide (Odavic et al. (1980) Experienta 36:1122–1124), and mouse marrow stem cells have been shown to be preserved by polyvinylpyrolidone (PVP), sucrose or glycerol. See, e.g., Stiff et al. (1987) Blood, 70(4): 974–978; Venkataraman, (1997) Cryobiology, 34:276–283; Wang et al., (1998) Cryobiology, 37:22–29; Merten et al., (1995) Biologicals, 23:185–189; and, Yoshida and Takeuchi, (1991) Cytotechnology 5:99–106. Polymers, such as hydroxyethyl starch (HES), have been used to cryoprotect human monocytes and unfractionated cells for use in bone marrow transplantation. See, e.g., Takashi et al., (1988) Biophysical Journal, 54:509–518; and Stiff et al. (1987) Blood, 70(4): 974–978.
Cryoprotective media for cellular vaccines has not been reported. Thus, there remains a need for cryoprotective media and procedures that can be used to successfully preserve cellular vaccines for use as therapeutic agents. In view of the above, materials and methods that would provide for successful preservation and recovery of cells with a high percentage viability following freezing and thawing would be highly desirable for use in cellular vaccines. The present invention addresses this need, as will be apparent from the detailed description provided herein.